Project Summary/Abstract Approximately 10,000 live births have defects in the craniofacial region in the United States annually. Severe craniofacial defects can result in lethality while less severe defects lead to morbidity and most surgical treatments only result in partial recovery. Identifying signaling factors that regulate cell fate decisions in craniofacial development is necessary for understanding the etiology of birth defects and identification of therapeutic targets. The Wnt/?-catenin signaling pathway functions in cell fate selection during development and cancer. Our lab has demonstrated that loss of Wnt/?-catenin signaling in the cranial mesenchyme results in a cell fate switch from skull bone to cartilage. How Wnt signaling promotes bone and dermal fate while inhibiting alternative cell fates in the cranial mesenchyme remains unclear. Previous studies have demonstrated that Erk1/2 activation is required for endochondral bone formation and growth as well as repression of cartilage differentiation during embryonic development. My preliminary data suggests that phosphorylation of Erk1/2 is diminished in the absence of ?-catenin in cranial mesenchyme at E12.5. I hypothesize that Wnt/?-catenin signaling represses cartilage and promotes skull bone formation through activation of Erk1/2 signaling in mice. To test this hypothesis I propose the following aims. 1) Determine if loss of phospho-Erk1/2 promotes cranial mesenchyme differentiation to cartilage. Here I propose to utilize two methods to inhibit Erk signaling in vivo, including genetic loss of Erk1/2 using En1Cre and chemical inhibition of Erk1/2 phosphorylation and assay for ectopic cartilage formation in the cranial mesenchyme at E12.5. Additionally I will overexpress Erk1/2 activation using a CA-Mek lentiviral infection in En1Cre;?-catfl/del calvarial mesenchyme in vitro. 2) Test whether phosphorylation of Erk1/2 is dependent on ?-catenin in the cranial mesenchyme. First I will demonstrate that loss of ?-catenin in cranial mesenchyme results in diminished Erk1/2 phosphorylation and determine whether this effect is due to Wnt signaling. Additionally, genetic interaction of ?-catenin and Erk1/2 will be determined by crossing En1Cre;?- catfl/del and Erk1-/-; Erk2fl/fl mice to obtain triple heterozygous loss-of-function embryos. Demonstrating a functional link between these two signaling pathways in regulating skull bone cell fate decisions in vivo will improve our understanding of normal skull bone development and associated birth defects, and more broadly, provide new avenues for bone repair in adult tissues. Successful completion of this proposal will provide insights into how Wnt/?-catenin signaling regulates cell fate decisions during cranial bone development and can be applied towards cell fate decisions in multiple cell types. Identifying new targets of Wnt signaling in bone development and cell fate could provide options for new therapeutic targets or preventative treatments of craniofacial disorders and would be applicable to a larger community that studies Wnt signaling and cell fate in other disease contexts, including cancer.